Clathrin-mediated endocytosis (CME) is a conserved process responsible for the selective uptake of nutrients and plasma membrane components. CME is a crucial way for cells to interact with, and react to, their external environments. Aberrant CME is implicated in many disease states, including atherosclerosis and diabetes. Additionally, a number of viruses enter the cell by endocytosis, and defects in endocytosis contribute to the metastatic potential of tumors by affecting cell polarity, proliferation, and migration. Understanding the mechanisms behind endocytosis is thus crucial for understanding normal cell function and pathogenesis. The budding yeast Saccharomyces cerevisiae makes an ideal system for studying CME, due to advantages such as easy genetic manipulation, low gene redundancy, and the existence of only one major endocytosis pathway. CME in yeast involves the ordered membrane recruitment, activity, and disassembly of ~60 endocytic proteins. Previous work in yeast has established the precise order of each protein's appearance at the plasma membrane, and almost all of this information has proven directly applicable to more complex organisms. However, the mechanism by which many of these proteins are recruited to the plasma membrane, particularly during endocytic site initiation, is still poorly understood. The goal of this research is to determine how endocytic membrane proteins are recruited to the plasma membrane. Speci?cally, this work will de?ne the role of Ede1 ubiquitination in downstream protein recruitment and will identify proteins that recruit Las17 to the plasma membrane. Actin tail assembly can be reconstituted on microbeads and synthetic membranes. Extending this work, endocytosis will be reconstituted on synthetic membranes coated with Ede1, an endocytic protein critical for site initiation. Ede1-coated membranes will be placed in cell extracts from yeast expressing tagged endocytic proteins. Primary outcomes used to con?rm successful reconstitution will include endocytic protein recruitment to membranes, actin tail formation, and vesicle release. To test the hypothesis that Ede1 ubiquitination effects downstream protein recruitment, mass spectrometry will be used to compare protein recruitment to membranes containing either Ede1 or ubiquitinated Ede1. To test the hypothesis that at least one upstream protein is a scaffold for Las17 recruitment, the above in vitro assay and an innovative strategy in live cells will be used. A fusion protein between an early (Sla2) and a late (Las17) endocytic protein will be expressed in yeast. This construct is expected to rescue las17 knockout cells and obviate the need for intermediate proteins that serve primarily as scaffolds. These two approaches promise to develop synergistically to identify mechanisms involved in endocytic site initiation and stabilization and thus increase our understanding of CME.